Molecular docking of four beta-amyloid1-42 fragments on the alpha7 nicotinic receptor: delineating the binding site of the Abeta peptides

Three-dimensional structures of the complexes between the Abeta(1-42) fragments Abeta(1-11), Abeta(10-20), Abeta(12-28), and Abeta(22-35) and the alpha7 nicotinic receptor were obtained with the aid of the ESCHER program. Furthermore, short high-temperature molecular dynamics simulations in vacuo were employed to relax the complexes and allow the peptides to accommodate in the binding site. The final models have shown that Abeta peptides do bind on the same site, which is delineated by loop C of one subunit and the loops 62-74 and G of the adjacent subunit on the receptor. This finding is supported by previous experimental and theoretical data, and should help one to obtain a better and more detailed structural information about the activity of the Abeta peptides and their repercussion in the disorders at molecular level, which are characteristic of the Alzheimer's disease.     

Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease.

The extracellular accumulation of amyloid-beta (Abeta) in neuritic plaques is one of the characteristic hallmarks of Alzheimer's disease (AD), a progressive dementing neurodegenerative disorder of the elderly. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteins and proteoglycans. The binding of the various forms of Abeta (soluble or fibrillar) to plasma membranes has been studied with regard to the direct toxicity of Abeta to neurons, and the activation of a local inflammation phase involving microglia. The binding of Abeta to membrane lipids facilitates Abeta fibrillation, which in turn disturbs the structure and function of the membranes, such as membrane fluidity or the formation of ion channels. A subset of membrane proteins binds Abeta. The serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind the monomeric form of Abeta. The alpha7nicotinic acetylcholine receptor (alpha7nAChR), integrins, RAGE (receptor for advanced glycosylation end-products) and FPRL1 (formyl peptide receptor-like 1) are able to bind the monomeric and fibrillar forms of Abeta. In addition, APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), the P75 neurotrophin receptor (P75NTR), the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha6beta1-integrin and CD47 have been reported to bind the fibrillar form of Abeta. Heparan sulfate proteoglycans have also been described as cell-surface binding sites for Abeta. The various effects of Abeta binding to these membrane molecules are discussed.     

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.     

Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors

Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.     

The alpha7 nicotinic acetylcholine receptor: molecular modelling, electrostatics, and energetics

The structure of a homopentameric alpha7 nicotinic acetylcholine receptor is modelled by combining structural information from two sources: the X-ray structure of a water soluble acetylcholine binding protein from Lymnea stagnalis, and the electron microscopy derived structure of the transmembrane domain of the Torpedo nicotinic receptor. The alpha7 nicotinic receptor model is generated by simultaneously optimising: (i) chain connectivity, (ii) avoidance of stereochemically unfavourable contacts, and (iii) contact between the beta1-beta2 and M2-M3 loops that have been suggested to be involved in transmission of conformational change between the extracellular and transmembrane domains. A Gaussian network model was used to predict patterns of residue mobility in the alpha7 model. The results of these calculations suggested a flexibility gradient along the transmembrane domain, with the extracellular end of the domain more flexible that the intracellular end. Poisson-Boltzmann (PB) energy calculations and atomistic (molecular dynamics) simulations were used to estimate the free energy profile of a Na+ ion as a function of position along the axis of the pore-lining M2 helix bundle of the transmembrane domain. Both types of calculation suggested a significant energy barrier to exist in the centre of the (closed) pore, consistent with a "hydrophobic gating" model. Estimations of the PB energy profile as a function of ionic strength suggest a role of the extracellular domain in determining the cation selectivity of the alpha7 nicotinic receptor. These studies illustrate how molecular models of members of the nicotinic receptor superfamily of channels may be used to study structure-function relationships.     

Peptide blockers of the inhibition of neuronal nicotinic acetylcholine receptors by amyloid beta

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.     

beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology

Alzheimer's disease pathology is characterized by the presence of neuritic plaques and the loss of cholinergic neurons in the brain. The underlying mechanisms leading to these events are unclear, but the 42-amino acid beta-amyloid peptide (Abeta(1-42)) is involved. Immunohistochemical studies on human sporadic Alzheimer's disease brains demonstrate that Abeta(1-42) and a neuronal pentameric cation channel, the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), are both present in neuritic plaques and co-localize in individual cortical neurons. Using human brain tissues and cells that overexpress either alpha7nAChR or amyloid precursor protein as the starting material, Abeta(1-42) and alpha7nAChR can be co-immunoprecipitated by the respective specific antibodies, suggesting that they are tightly associated. The formation of the alpha7nAChR.Abeta(1-42) complex can be efficiently suppressed by Abeta(12-28), implying that this Abeta sequence region contains the binding epitope. Receptor binding experiments show that Abeta(1-42) and alpha7nAChR bind with high affinity, and this interaction can be inhibited by alpha7nAChR ligands. Human neuroblastoma cells overexpressing alpha7nAChR are readily killed by Abeta(1-42), whereas alpha7nAChR agonists such as nicotine and epibatidine offered protection. Because Abeta(1-42) inhibits alpha7nAChR-dependent calcium activation and acetylcholine release, two processes critically involved in memory and cognitive functions, and the distribution of alpha7nAChR correlates with neuritic plaques in Alzheimer's disease brains, we propose that interaction of the alpha7nAChR and Abeta(1-42) is a pivotal mechanism involved in the pathophysiology of Alzheimer's disease.     

The beta-amyloid protein of Alzheimer's disease binds to membrane lipids but does not bind to the alpha7 nicotinic acetylcholine receptor

Accumulation of the amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Abeta exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In this study, we examined the binding of Abeta1-42 to endogenous and recombinantly expressed alpha7nAChRs. Abeta1-42 did neither inhibit the specific binding of alpha7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of alpha7nAChRs expressed in Xenopus oocytes. Similarly, Abeta1-42 did not compete for alpha-bungarotoxin-binding sites on SH-SY5Y cells stably expressing alpha7nAChRs. The effect of the Abeta1-42 on tau phosphorylation was also examined. Although Abeta1-42 altered tau phosphorylation in alpha7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to alpha7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Abeta bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Abeta may disrupt membrane lipid structure or fluidity. We conclude that the effects of Abeta are unlikely to be mediated by direct binding to the alpha7nAChR. Instead, we speculate that Abeta may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.     

Differential physiologic responses of alpha7 nicotinic acetylcholine receptors to beta-amyloid1-40 and beta-amyloid1-42

The beta-amyloid peptides (Abeta), Abeta(1-40) and Abeta(1-42), have been implicated in Alzheimer's disease (AD) pathology. Although Abeta(1-42) is generally considered to be the pathological peptide in AD, both Abeta(1-40) and Abeta(1-42) have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Abeta peptides, when interact with the neuronal cation channel, alpha7 nicotinic acetylcholine receptors (alpha7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Abeta(1-42) effectively attenuated these alpha7nAChR-dependent physiology to an extent that was apparently irreversible, Abeta(1-40) showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with alpha7nAChR antagonists. Our data suggest a clear pharmacological distinction between Abeta(1-40) and Abeta(1-42).     

Solution conformation of alpha-conotoxin EI, a neuromuscular toxin specific for the alpha 1/delta subunit interface of torpedo nicotinic acetylcholine receptor

A high resolution structure of alpha-conotoxin EI has been determined by (1)H NMR spectroscopy and molecular modeling. alpha-Conotoxin EI has the same disulfide framework as alpha 4/7 conotoxins targeting neuronal nicotinic acetylcholine receptors but antagonizes the neuromuscular receptor as do the alpha 3/5 and alpha A conotoxins. The unique binding preference of alpha-conotoxin EI to the alpha(1)/delta subunit interface of Torpedo neuromuscular receptor makes it a valuable structural template for superposition of various alpha-conotoxins possessing distinct receptor subtype specificities. Structural comparison of alpha-conotoxin EI with the gamma-subunit favoring alpha-conotoxin GI suggests that the Torpedo delta-subunit preference of the former originates from its second loop. Superposition of three-dimensional structures of seven alpha-conotoxins reveals that the estimated size of the toxin-binding pocket in nicotinic acetylcholine receptor is approximately 20 A (height) x 20 A (width) x 15 A (thickness).     

Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.     

An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced alpha4 residues into the alpha7/5HT(3) chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch-clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-affinity alpha-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric-kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in alpha4 and in alpha7 double mutant but not in alpha7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.     

Solution structure of alpha-conotoxin PIA, a novel antagonist of alpha6 subunit containing nicotinic acetylcholine receptors

alpha-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing alpha6 and alpha3 subunits. alpha-conotoxin PIA displays 75-fold higher affinity for rat alpha6/alpha3beta2beta3 nAChRs than for rat alpha3beta2 nAChRs. We have determined the three-dimensional structure of alpha-conotoxin PIA by nuclear magnetic resonance spectroscopy. The alpha-conotoxin PIA has an "omega-shaped" overall topology as other alpha4/7 subfamily conotoxins. Yet, unlike other neuronally targeted alpha4/7-conotoxins, its N-terminal tail Arg1-Asp2-Pro3 protrudes out of its main molecular body because Asp2-Pro3-Cys4-Cys5 forms a stable type I beta-turn. In addition, a kink introduced by Pro15 in the second loop of this toxin provides a distinct steric and electrostatic environment from those in alpha-conotoxins MII and GIC. By comparing the structure of alpha-conotoxin PIA with other functionally related alpha-conotoxins we suggest structural features in alpha-conotoxin PIA that may be associated with its unique receptor recognition profile.     

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

Primary structure and expression of beta 2: a novel subunit of neuronal nicotinic acetylcholine receptors

A new subunit, beta 2, of the neuronal nicotinic receptor family has been identified. This subunit has the structural features of a non-agonist-binding subunit. We provide evidence that beta 2 can substitute for the muscle beta 1 subunit to form a functional nicotinic receptor in Xenopus oocytes. Expression studies performed in oocytes have demonstrated that three different neuronal nicotinic acetylcholine receptors can be formed by the pairwise injection of beta 2 mRNA and each of the neuronal alpha subunit mRNAs. The beta 2 gene is expressed in PC12 cells and in areas of the central nervous system where the alpha 2, alpha 3, and alpha 4 genes are expressed. These results lead us to propose that the nervous system expresses diverse forms of neuronal nicotinic acetylcholine receptors by combining beta 2 subunits with different agonist-binding alpha subunits.     

NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha-subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 versus alpha6beta2, and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2, and alpha1beta1deltaepsilon nAChRs.     

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.